Although effective neuraminidase inhibitors are available to treat influenza viral infections in humans, the emergence of virus isolates resistant to these drugs can have a significant adverse impact on both treatment options and disease outcome. To better prepare us to cope with an emerging influenza pandemic and combat the rising problem of antiviral drug resistance, new classes of antiviral drugs targeted to conserved viral functions are urgently needed. One such target for anti-influenza drug development is the assembly of the viral RNA polymerase complex. The viral RNA polymerase complex (PA-PB1-PB2) is a heterotrimer composed by the three subunits, polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). PB1 subunit forms the core of the polymerase complex. PB1 interacts through its N-terminal region with the C- terminal region of PA, while the C-terminal region of PB1 is involved in an interaction with PB2. No interaction between PA and PB2 subunits has been reported. While the molecular mechanism of the polymerase complex formation remains unclear, the assembly of viral RNA polymerase complex during viral replication is a highly regulated and dynamic process and is essential for viral RNA synthesis and production of infectious influenza virus particles. Any disruption of the viral RNA polymerase complex formation or even inhibition of sequential nature of the assembly process profoundly impairs the transcription and translation of viral RNA segments and viral infectivity. Proof of principle for inhibition of influenza A virus RNA polymerase complex assembly has been recently demonstrated by developing a peptide-based inhibitor which potently inhibits growth of influenza A viruses through specifically blocking the PB1-PA interaction and subsequently interfering with viral RNA polymerase complex assembly. These studies support our focus on influenza A RNA polymerase formation as a target for therapeutic discovery and development. The overall goal of this research proposal is to develop a novel assay that may lead to the identification of antiviral compounds, which inhibit Influenza A virus replication by disrupting the PB1-PA subunit interaction that is critical to govern the assembly process of influenza A virus RNA polymerase complex. The identification of specific influenza virus inhibitors will serve two major purposes. First, these compounds will provide valuable tools for dissecting distinct stages of the assembly process of viral RNA polymerase complex at the cellular and molecular level. Second, some of the identified compounds may serve as lead compounds for the development of novel anti-influenza drugs. The technical foundation for the proposed assay is the use of Bimolecular Fluorescence Complementation (BiFC) which is based on the principle that N- and C-terminal fragments of fluorescent proteins (GFP and its derivatives) do not spontaneously fold and reconstitute a functional fluorophore. However, if fused to interacting proteins, the two non-functional halves of the fluorophore, following the expression in cells, are brought into close proximity as a result of the specific protein interactions. This initiates folding of the fragments into an active protein, which then can reconstitute a detectable fluorescent signal at the site of the protein-protein complex. Thus, through BiFC, the specific interaction between PB1 and PA subunits can be precisely visualized, quantified and localized within live cells. By disrupting PB1-PA interaction, compounds will cause reductions in BiFC readout, indicative of the presence of potential inhibitors targeting the assembly of PB1-PA complex. Two specific aims are proposed in the application: (1) to develop and characterize BiFC constructs which allow for visual identification of PB1-PA dimeric complex in living cells, and (2) to develop a BiFC-based prototypic influenza A virus RNA polymerase complex assembly inhibitor screening assay suitable for use in a high-throughput format. It is anticipated that this research project will establish a proof-of-concept for the BiFC approach to identifying assembly inhibitors of influenza A virus RNA polymerase complex, which will provide the basis for the development of a high-throughput screening assay. Importantly, successful accomplishment of this project will lead to the validation of a BiFC-based approach to drug screening for viral pathogens. This could then be applied to NIAID category A, B or C viral agents, which could not be carried out in most locations because of biosafety concerns. PUBLIC HEALTH RELEVANCE Drug resistance poses a great challenge for anti-influenza therapy and contributes to influenza treatment failure. Successful completion of the proposed research will help identify and design additional anti-influenza inhibitors that will provide additional treatment options and improve disease outcome.